Principles of Fluorescence Spectroscopy

514 ENERGY TRANSFER TO MULTIPLE ACCEPTORS IN ONE,TWO, OR THREE DIMENSIONS
Figure 15.9. Rhodamine 6G donor decays absorbed to dihexadecyl
phosphate vesicles. The acceptor was malachite green with concentrations
of 0 M (1), 3.32 x 10 –6 M (2), 4.40 x 10 –6 M (3), and 5.27 x 10 –6
M (4). Revised and reprinted with permission from [23]. Copyright ©
1987, American Chemical Society.
sions. Energy transfer to surface-localized fluorophores on
the side of the membrane is likely to be two dimensional.
However, RET may display a different dimensionality for
donors on the surface transferring to acceptors dispersed in
the acyl side chain region. Also, acceptors on the opposite
surface from the donor may contribute a three-dimensional
component to the donor decay. While such systems can be
interpreted in terms of fractal dimensions, the concept of
fractal dimensions is rather abstract and does not always
lead to physical insights. In these cases it is useful to consider
a combination of energy transfer in two and three
dimensions.
Energy transfer with a mixed dimensionality was found
for the time-resolved donor decays from rhodamine 6G to
malachite green, when both were bound to vesicles of
dihexadecyl phosphate (DHP). 23 The donor decays are
shown in Figure 15.9. In this case it was necessary to fit the
data to a sum of eqs. 15.1 and 15.9. Although not shown,
neither eqs. 15.1 or 15.9 alone provided a good fit to these
data. While the authors interpreted this effect in terms of a
non-random acceptor distribution, energy transfer across
the bilayer could also have provided a component that
appeared to be three dimensional. Once again, comparison
of the steady-state data with predicted donor intensities was
essential for selecting between distinct models. 23
15.2.2. Experimental FRET in One Dimension
While there have been numerous studies of the timeresolved
fluorescence of dyes bound to DNA, 31 there have
been relatively few studies of RET for dyes intercalated into
Figure 15.10. Emission spectra of dimethyldiazaperopyrenium bound
to poly d(A-T) with increasing concentrations of ethidium bromide.
The insert shows a comparison of the measured (!) and predicted (")
transfer efficiencies. Revised from [32].
DNA. 32–35 In Figure 15.6 we showed simulations that indicated
a rapidly decaying t 1/6 component for donors and
acceptors in one dimension. Such time-dependent decays
have been observed with dyes intercalated into poly d(A-
T). 32 The donor was dimethyldiazaperopyrenium (DMPP)
and the acceptor ethidium bromide (EB). Upon binding of
the EB acceptor the DMPP emission was quenched, and the
EB emission was enhanced (Figure 15.10). The timedomain
data clearly show a fast component with increasing
amplitude as the acceptor concentration is increased (Figure
15.11). Due to a lack of software the time-domain data were
not analyzed in terms of eq. 15.12, but the shape of the
decays is visually similar to the simulated data for RET in
one dimension (Figure 15.6).
Useful information can also be obtained by examination
of the steady-state data. For the D–A pair the observed
transfer efficiency (Figure 15.10, insert) was found to be
smaller than that predicted from Monte Carlo simulations.
This result was explained as distortion of the DNA by binding
of DMPP, which excluded EB from binding to nearby
sites.
Energy transfer in one dimension was also studied
using the frequency-domain method. 18 In this case the
donor was acridine orange (AO) and the acceptor was the
weakly fluorescent dye Nile blue (NB). Binding of NB at a
low dye-per-base-pair ratio results in significant quenching
of the AO emission (Figure 15.12). The FD intensity decay
data are best fit by the equation for FRET in one dimension
(Figure 15.13), indicating that energy transfer occurs in one
dimension in this system.